Sorghum (Sorghum biocolor (L.) Moench) is recognized as a low-input forage and bioenergy crop that is highly resilient to the effects of climate change. The crop generally requires about half the water to grow compared to corn and exhibits excellent adaptation to low-input production systems. Given these characteristics, it is one of the most highly-touted of the new bioenergy crops identified for use in future biomass production systems.
Sorghum produces a large number of secondary metabolites including the cyanogenic glucoside dhurrin. Cyanogenic glucosides belong to a class of plant-produced antibiotics called phytoanticipins (Vanetten et al., 1994; Osbourn et al., 1996). More than 2,650 plant species are cyanogenic and able to release HCN upon tissue disruption (Conn, 1981; Siegler and Brinker, 1993) including pteridophytes, gymnosperms and angiosperms. Phylogenetic analyses suggest the evolution of cyanogenesis occurred before the bifurcation of pteridophytes and gymnosperms. Cyanogenic glucosides are particularly important in crop plants used for animal and human nutrition such as sorghum, barley [Hordium vulgare L.], and cassava [Manihot esculenta Crantz].
Biochemical studies have shown that plant cyanogenic glucosides are derived from six different amino acids: the aliphatic protein amino acids valine, isoleucine and leucine; the aromatic amino acids phenylalanine and tyrosine; and the aliphatic nonproteinogenic amino acid cyclopentenyl glycine (Jones, 2000; Jaroszewski et al., 2002). Fern and gymnosperm species generally produce cyanogenic glucosides from the aromatic amino acids, whereas angiosperms produce cyanogenic glucosides from aliphatic and aromatic amino acids (Bak et al., 2006). Arthropods including millipedes (Diploda), centipedes (Chilopoda), beetles (Coleoptera), and true bugs (Heteroptera) also produce cyanogenic glucosides that are synthesized from aromatic amino acids or in the case of lepidopterans from aliphatic amino acids (Bak et al., 2006; Zagrobelny et al., 2008).
When plant tissues are disrupted through chewing or tissue maceration, the cyanogenic glucosides in the vacuoles are brought into contact with β-glucosidases and α-hydroxynitrile lyases that hydrolyze the cyanogenic glucosides and produce HCN (Vetter et al., 2000). This HCN renders the consumption of plant materials containing cyanogenic glucosides toxic to humans and most animals (Oluwole et al., 2000). The affinity of cyanide for the terminal cytochrome oxidase in the mitochondrial respiratory pathway is the main cause of toxicity (Brattsten et al., 1983). A lethal dose of cyanide for vertebrate animals is in the range of 35-150 μmol kg-1, but higher amounts can be tolerated if consumed over a longer period (Davis and Nahrstedt, 1985). Studies in horses showed that sorghum-based diets consumed over a 2-month period resulted in incoordination of the hind legs, urinary incontinence, and haematuria. These symptoms were followed by increased nasal discharge, increased body temperature, and depression of appetite (Varshney et al., 1996).
Given the toxicity of HCN, cyanogenic glucosides are assumed to play a role in plant defense against animal and insect herbivores. The effects of cyanogenic plants in human and animal health is well documented (Banea-Mayambu et al., 1997, 2000; Robinson, 1930; Boyd, 1938; Webber et al., 1985; Hopkins et al., 1995). In the case of sorghum forage, cyanide poisoning resulting in cattle death was first reported in Australia more than a hundred years ago (Anon, 1897).
Sorghum was used as the model to dissect the cyanogenic glucoside biosynthetic pathway in plants. All of the structural genes in this pathway have been cloned (Jones et al., 2000) (FIG. 1). The S. bicolor genome was sequenced and is available from the Phytozome project, which is a joint project of the Department of Energy's Joint Genome Institute and the Center for Integrative Genomics, at http://www.phytozome.net/sorghum. The sequenced genome consists of 697,578,683 base pairs arranged in 2n=20 chromosomes. It has 34,496 loci containing protein-coding transcripts and 36,338 protein-coding transcripts. The S. bicolor genome was published in Paterson et al. 2009 and will be referred to herein by its identifier Tx623.
Dhurrin is a cyanogenic glucoside of sorghum and dhurrin accumulation in plant tissues negatively impacts forage and feedstock quality. Sorghum breeders are working to modify dhurrin content to improve forage and biomass quality but are constrained by a lack of natural genetic variation for this trait in the elite sorghum gene pool. Mutation breeding is being used to induce mutations in the dhurrin biosynthesis pathways. One mutant was recently identified in Australia that produced no dhurrin in any tissue but exhibited “slightly slower growth at early seedling stage.” (Blomstedt, 2012). Different mutations or alleles that similarly disrupt dhurrin biosynthesis but without impacts on growth may be better suited to commercial product development.
Dhurrin content is highest in young seedlings and can represent as much as 6 to 10% of the dry weight of plants (Akazawa et al., 1960; Conn, 1994). Accumulations as high as 30% dry weight have been reported in some parts of sorghum seedlings (Halkier et al., 1989). However, little is known about the downstream processing and utilization of cyanogenic glucosides in sorghum or any other plant species.
Thus, there is a need for a low-dhurrin or dhurrin-free sorghum plant that has a standard growth rate compared to existing commercial varieties.